The future of 5-HT1A receptor agonists. (Aryl-piperazine derivatives).

Mitsukuni Murasaki and Sadanori Miura: The Future of 5-HT_1A Receptor Agonists. (Aryl-Piperazine Derivatives) Prog. Neuro- Psychopharmacol-& Biol Psychiat, 1992, 16(6): 833–845.

1. 1. At present the dominant position among anti-anxiety medications has changed from meprobamate to the benzodiazepine derivatives.

2. 2. In order to avoid benzodiazepine's (BZ) undesirable side effects such as impairment of psycho-motor function, memory impairment, low dose dependence and withdrawal symptoms, a third generation anxiolytic agent, buspirone, the focus of the aryl-piperazine group of anti-anxiety agents, has been introduced recently.

3. 3. Aryl-piperazine derivatives work as 5-HT_1A receptor partial agonists and are known as serotonin normalizers.

4. 4. Therefore, they are expected to have not only an anxiolytic function but also an anti-depressant effect as well.

5. 5. A characteristic of the aryl-piperazine derivatives is that they have no sedative and muscle relaxant effects, and they do not have BZ's undesirable side-effects, especially in regard to withdrawal symptoms. However they have a rather weak anxiolytic action and a slow onset of action.

6. 6. Aryl-piperazine derivatives will not take the place of BZ, but the use of BZ and buspirone as bridge medications, making the most of the strong points of both, can be proposed as a way to compensate for their respective disadvantages.

Increased midbrain 5-HT1A receptor number and responsiveness in cholestatic rats

Midbrain somatodendritic 5-HT_1A autoreceptors play a central inhibitory role in the regulation of serotonergic neurotransmission. Given that serotonergic neurotransmission appears to be altered in experimental cholestatic liver disease we examined alterations in midbrain 5-HT_1A autoreceptor binding and physiological responses in rats with experimental cholestatic liver disease in comparison to non-cholestatic controls. Using a standard receptor binding assay cholestatic rats exhibited an increase in midbrain 5-HT_1A receptor number but no change in receptor affinity compared to controls. Midbrain 5-HT_1A receptor mRNA expression as determined by semiquantitative RT–PCR was similar in cholestatic and non-cholestatic animals. In addition, cholestatic rats exhibited enhanced 5-HT_1A autoreceptor-mediated hypothermic and hyperphagic responses compared to non-cholestatic controls after the administration of the highly specific 5-HT_1A receptor agonist LY293284. These findings indicate that experimental cholestatic liver injury is associated with enhanced 5-HT_1A autoreceptor-mediated physiological responsiveness in the setting of increased midbrain 5-HT_1A receptor number but not affinity.     

Serotonin neuron-dependent and -independent reduction of dyskinesia by 5-HT1A and 5-HT1B receptor agonists in the rat Parkinson model

5-HT₁ receptor agonists have been shown to reduce abnormal involuntary movements (AIMs) in the rat and monkey models of l-DOPA-induced dyskinesia. Different mechanisms have been proposed to underlie this effect. Activation of pre-synaptic 5-HT₁ receptors has been suggested to inhibit dysregulated release of dopamine from the serotonin terminals, and thus, abnormal activation of striatal dopamine receptors. Activation of post-synaptic 5-HT₁ receptors expressed in non-serotonergic neurons in different brain areas, by contrast, has been shown to result in decreased glutamate and GABA release, which may also contribute to the antidyskinetic effect.To unveil the relative contribution of these mechanisms, we have investigated the effect of increasing doses of 5-HT_1A and 5-HT_1B receptor agonists on AIMs induced by either l-DOPA or apomorphine. In contrast to l-DOPA-induced AIMs, which were dampened already at low doses of 5-HT₁ agonists, reduction of apomorphine-induced AIMs required higher doses. Removal of the serotonin innervation suppressed l-DOPA-induced AIMs, but neither affected apomorphine-induced AIMs nor the inhibiting effect of 5-HT₁ agonists on AIMs induced by the direct dopamine agonist, suggesting that such effect is independent on activation of pre-synaptic 5-HT₁ receptors.     

Effects of triiodothyronine and fluoxetine on 5-HT1A and 5-HT1B autoreceptor activity in rat brain: regional differences

The thyroid hormone triiodothyronine (T3) augments and accelerates the effects of antidepressant drugs. Although the majority of studies showing this have used tricyclics, a few studies have shown similar effects with the selective serotonin re-uptake inhibitor (SSRI) fluoxetine. In this study we investigated the effects of fluoxetine (5 mg/kg), T3 (20 μg/kg) and the combination of these drugs, each administered daily for 7 days, on serotonergic function in the rat brain, using in vivo microdialysis. Fluoxetine alone induced a trend towards desensitization of 5-HT_1A autoreceptors as shown by a reduction in the effect of 8-OH-DPAT to lower 5-HT levels in frontal cortex, and desensitized 5-HT_1B autoreceptors in frontal cortex. The combination of fluoxetine and T3 induced desensitization of 5-HT_1B autoreceptors in hypothalamus. Since there is evidence linking hypothalamic function and depression, we suggest that this effect may partly account for the therapeutic efficacy of the combination of an SSRI and T3.     

Effects of adrenalectomy and type I or type II glucocorticoid receptor activation on 5-HT1A and 5-HT2 receptor binding and 5-HT transporter mRNA expression in rat brain

We evaluated the effects of adrenalectomy (ADX) and replacement with glucocorticoid receptor agonists on serotonin (5-HT) 5-HT_1A and 5-HT₂ receptor binding in rat brain. 5-HT_1A receptor binding was increased in the CA2–CA4 and the dentate gyrus of the hippocampus 1 week after ADX. This effect was prevented by the systemic administration of aldosterone (10 μg/μl/h) but not by RU28362 (10 μg/μl/h). No significant effect was observed on 5-HT₂ receptor binding in rat cortex. The expression of 5-HT transporter mRNA was unchanged in the raphe nucleus as measured by in situ hybridization.     

5-HT1A and 5-HT2 receptors differentially regulate the excitability of 5-HT-containing neurones of the guinea pig dorsal raphe nucleus in vitro

We have used intracellular recording techniques to examine the effects of 5-hydroxytryptamine (5-HT, serotonin) on 5-HT-containing neurones of the guinea pig dorsal raphe nucleus in vitro. Bath-applied 5-HT (30–300 μM) had two opposing effects on the membrane excitability of these cells, reflecting the activation of distinct 5-HT receptor subtypes. As demonstrated previously in the rat, 5-HT evoked a hyperpolarization and inhibition of 5-HT neurones, which appeared to involve the activation of an inwardly rectifying K⁺ conductance. This hyperpolarizing response was blocked by the 5-HT_1A receptor-selective antagonist WAY-100635 (30–100 nM). In the presence of WAY-100635, 5-HT induced a previously unreported depolarizing, excitatory response of these cells, which was often associated with an increase in the apparent input resistance of the neurone, likely due to the suppression of a K⁺ conductance. Like the hyperpolarizing response to 5-HT, this depolarization could be recorded in the presence of the Na⁺ channel blocker tetrodotoxin. In addition, the response was not significantly attenuated by the α₁-adrenoceptor antagonist prazosin (500 nM), indicating that it is not due to the release of noradrenaline, or to the direct activation of α₁-adrenoceptors by 5-HT. The 5-HT₃ receptor antagonist granisetron (1 μM) and the 5-HT₄ receptor antagonist SB 204070 (100 nM) failed to reduce the depolarizing response to 5-HT; however, ketanserin (100 nM), mesulergine (100 nM) and lysergic acid diethylamide (1 μM) significantly reduced or abolished the depolarization, indicating that this effect of 5-HT is mediated by 5-HT₂ receptors.     

Indications of pre- and post-synaptic 5-HT1A receptor interactions in feeding behavior and neuroendocrine regulation

This bipartite study uses behavioral and biochemical means to explore the involvement of both pre- and post-synaptic 5-HT_1A receptors in the control of food intake and neuroendocrine regulation. In the pharmacological study, the administration of the 5-HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT; 60 μg/kg b.wt., i.p.) to rats caused a significant increase in 2 h intake of a high carbohydrate (CARB)/sugar diet (P < 0.05) during the relatively inactive feeding period of the late light cycle. No significant change was detected in the intake of Purina laboratory chow at 2 h, or of the intake of either diet at 4 h and 24 h after 8-OH-DPAT administration. Injection of 8-OH-DPAT induced a drop in insulin levels in rats maintained on high CARB/sugar diets only (−90%; P<0.05). It also caused an increase in circulating glucose levels in both high CARB/sugar (240%; P<0.01) and chow fed (123%; P<0.05) rats; it did so more intensely in high CARB/sugar-fed rats. In the biochemical study, radioligand binding techniques were used to assess 5-HT_1A receptor density in the hypothalamus, as well as the relationship between 5-HT_1A receptors and circulating levels of insulin and glucose. Chronic and acute administration (25 mg/kg b.wt./5 injections, and 50 mg/kg b.wt., respectively, i.p.) of the potent hypoglyce mic agent tolbutamide (TOL) caused a significant increase in 5-HT_1A receptor density (+243% and +132.6%, respectively; P<0.05) in the medial hypothalamus but not in the lateral hypothalamus, as compared to vehicle-treated rats. Chronic glucose replacement therapy showed a trend towards reversing the depressed circulating glucose levels as well as the medial hypothalamic 5-HT_1A receptor density to control levels. These studies indicate that the pre-synaptic mechanism of 8-OH-DPAT-induced hyperphagia may require specific circulating levels of insulin and glucose, which are regulated via post-synaptic 5-HT_1A receptors.     

Stimulation of 5-HT1A receptors reduces apoptosis after transient forebrain ischemia in the rat

It has recently been shown that 5-HT_1A receptor stimulation reduced the infarct volume after occlusion of the middle cerebral artery in rats. Since there is increasing evidence that apoptosis is involved in neurodegenerative diseases and stroke, we investigated whether the 5-HT_1A agonist Bay x 3702 could protect neurons against apoptotic damage in a rat model of transient forebrain cerebral ischemia. Bay x 3702 (4 μg/kg i.v.) caused a 10% reduction of neuronal damage in the hippocampal CA1 subfield. Higher doses of Bay x 3702 (40 and 12 μg/kg i.v.) did not cause any neuroprotective effect, most likely because of the strong reduction of mean arterial blood pressure during the period of Bay x 3702 infusion. Bay x 3702 (4 μg/kg i.v.) diminished DNA laddering in the hippocampus and striatum 4 days after 10 min forebrain ischemia. These results were confirmed by TUNEL-staining. The anti-apoptotic effect was abolished by additional treatment with the 5-HT_1A receptor antagonist WAY 100635 (1 mg/kg). Taken together, the results suggest that Bay x 3702 can rescue hippocampal as well as striatal neurons from apoptotic cell death in vivo via stimulation of 5-HT_1A receptors.     

Sleep/waking effects following intrathecal administration of the 5-HT1A agonist 8-OH-DPAT alone and in combination with the putative 5-HT1A antagonist NAN-190 in rats

Sleep, waking, and EEG power spectra were investigated in rats after intrathecal (IT) administration of a 5-HT_1A agonist and a 5-HT_1A antagonist. Total slow wave sleep (TSWS) was increased and waking was decreased over the 8-h recording period after the 5-HT_1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (38 nmol). Within TSWS, SWS1 was unchanged while SWS-2 tended to be increased. The 5-HT_1A antagonist 1-[2-Methoxyphenyl)-4-(4-(2-phthalimido)-butyl]piperazine hydrobomide (NAN-190) did not change and sleep/waking stages. Combined treatment with 8-OH-DPAT and NAN-190 increased variance. Following the combination, sleep and waking were not significantly different from control. SWS-2 tended to be reduced compared to the effect of 8-OH-DPAT alone. There were no systematic changes in neither waking nor TSWS fronto-frontal or fronto-parietal EEG power spectrum after any of the treatments, indicating that sleep quality was not changed. The results confirm earlier data suggesting that in the spinal cord, stimulation of 5-HT1A receptors have a dampening effect on transmission of sensory information, leading to deactivation and thereby increased sleep tendency. The reason why the 8-OH-DPAT effect was not clearly antagonized by the putative 5-HT1A antagonist NAN-190, may be due to the generally weak antagonistic and also partial agonistic effect of NAN-190 as reported in the literature.     

Selective labeling of 5-HT1A and 5-HT1B binding sites in bovine brain

Drug interactions with serotonin(1A) 5-HT1A and serotonin(1B) (5-HT1B) binding sites were analyzed in bovine brain membranes. 5-HT1A binding sites were directly labeled with [3H]8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) in bovine hippocampal membranes. 5-HT1B binding sites were labeled by [3H]5-HT in bovine striatal membranes where less than 15% of specific binding sites are sensitive to nanomolar concentrations of 8-OH-DPAT. Each of the 12 agents tested was more potent at the 5-HT1A than 5-HT1B binding site. 5-HT, bufotenine, N,N-dimethyltryptamine (DMT) and quipazine were only slightly more potent at the 5-HT1A binding site. By contrast, 8-OH-DPAT, TVX Q 7821 and buspirone were significantly more potent at [3H]8-OH-DPAT binding sites in bovine hippocampus than at [3H]5-HT binding sites in bovine striatum. These findings suggest that 5-HT1A, and 5-HT1B binding sites have distinct pharmacological profiles and can be directly labeled with appropriate [3H]ligands in specific brain regions.     

Protective effects of 5-HT1A receptor agonists against neuronal damage demonstrated in vivo and in vitro

Summary The aim of the present study was to evaluate the neuroprotective effect of the 5-hydroxytryptamine_1A (5-HT_1A) agnists, CM 57493 and urapidil, in vivo and in vitro, respectively. In vivo permanent occlusion of the middle cerebral artery (MCA) was performed in male Wistar rats. Forty-eight hours after electrocoagulation of the MCA and infarct volume was determined. Pretreatment of the rat with the 5-HT_1A agonist urapidil significantly reduced infarct development. The neuroprotective effect of the agent was restricted to the cortical area; the striatal damage was not influence. As the stimulation of the 5-HT_1A receptor by serotonin is supposed to induce inhibitory, hyperpolarizing effects by opening of a Ca²⁺-independent neuronal K⁺ ionophore, the efficacy of agonistic drugs directly on the neuron was investigated in vitro. Cyanide-induced cytotoxic hypoxia as well as glutamate-induced excitotoxicity were performedj using primary neuronal cell cultures from chick embryo cerebral hemispheres. Treatment with 5-HIT_1A agonists urapidil and CM 57493 significantly increase protein content of hypoxic cultures. CM 57493 and added to the culture medium (1–10 M) during and up to 24 h after glutamate exposure ameliorated viability of the neurons. The result demonstrate neuroprotective potency of the 5-HT_1A agonists, urapidil and CM 57493, when applied under hypoxic, excitotoxic and ischemic conditions in vivo and in vitro, respectively. Both, presynaptically induced inhibition of glutamate release as well as postsynaptically induced inhibition of neuronal excitability could be discussed as possible mechanisms of action of the 5-HT_1A receptor agonism.     

Implication of the 5-HT2A and 5-HT2C (but not 5HT1A) receptors located within the periaqueductal gray in the elevated plus-maze test–retest paradigm in mice

A single exposure to the elevated plus-maze test (EPM) increases open arms avoidance and reduces or abolishes the anxiolytic-like effect of benzodiazepines assessed during a second trial, a phenomenon defined as “one-trial tolerance” (OTT). It has been emphasized that the dorsal portion of the midbrain periaqueductal gray (dPAG) plays a role on this enhanced aversion phenomenon in maze-experienced rodents. Given that intra-dPAG injections of a wide range of serotonergic 5-HT_1A, 5-HT_2A and 5-HT_2C receptor agonists produce anxiolytic-like effects in maze-naïve rodents, the present study examined the effects of the 5-HT_1A receptor agonist 8-OH-DPAT (5.6 and 10.0 nmol in 0.15 µl) the preferential 5-HT_2A receptor agonist DOI (2.0 and 8.0 nmol in 0.1 µl) and the preferential 5-HT_2C receptor agonist MK-212 (21.2 and 63.6 nmol in 0.1 µl) microinjected into the dPAG prior to Trial 1 and Trial 2 on the behaviour of mice in the EPM. Test sessions were recorded and subsequently scored for anxiety-like behaviour (percentage of open arms entries and time) as well as general locomotor activity (closed arm entries). The results showed a lack of 8-OH-DPAT (5.6 and 10.0 nmol) effect on the behaviour of maze-naïve and maze-experienced mice, while intra-dPAG microinfusions of DOI (8 nmol) reduced anxiety-like behaviour only in maze-experienced mice that had received a similar treatment prior to Trial 1. Furthermore, intra-dPAG MK-212 (63.6 nmol) showed an anxiolytic-like effect on both Trial 1 and Trial 2. Importantly, these effects were observed in the absence of any significant change in closed arm entries, the parameter considered to be a valid index of locomotor activity in the plus-maze. These results support the dPAG as a crucial structure involved in the neurobiology of the OTT phenomenon as well as accounting the role of the 5-HT_2A and 5-HT_2C receptors located within this midbrain structure on the emotional state induced by EPM test and retest paradigm mice.     

Age-related assessment of central 5-HT1A receptors following irreversible inactivation by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)

Age-dependent differences in the ability of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) to irreversibly inactivate 5-HT_1A receptors were investigated in female Fischer 344 rats (ages 3 and 22 months). In the hippocampus, frontal cortex and amygdala, EEDQ reduced 5-HT_1A receptor density (33–70%) and drug affinity (2.3–6.2 fold) as determined by Scatchard analyses using [³H]8-hydroxy-2-(di-N-propylamino)tetralin. In the frontal cortex, the reduction in B_max values was significantly greater in 3 months vs. 22 months groups. These region-specific and age-dependent alterations in 5-HT_1A receptors may be of pathophysiological significance in age-related cognitive decline and Alzheimer's disease.     

Effects of 8-OH-DPAT, a 5-HT1A receptor agonist, and DOI, a 5-HT2A/2C agonist, on monosynaptic transmission in spinalized rats

We investigated the effect of the 5-HT_1A receptor agonist (±)-8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) and the 5-HT_2A/2C receptor agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) on monosynaptic transmission in spinalized rats. 8-OH-DPAT significantly inhibited the excitation of α-motoneurons evoked by monosynaptic transmission without a direct effect on α-motoneuron excitation. DOI potentiated the excitation of α-motoneurons by both direct stimulation and monosynaptic transmission. These results indicate that activation of 5-HT_1A receptors inhibits monosynaptic transmission, whereas activation of 5-HT_2A/2C receptors enhances it.     

Maternal deprivation increases 5-HT1A receptor expression in the CA1 and CA3 areas of senescent Brown Norway rats

Maternally-deprived male Brown Norway rats were classified as non-impaired or impaired according to their performance in the water maze when 3 and 30–32 months old. Age and spatial learning ability did not affect the pattern and density of hippocampal 5-HT_1A-receptor mRNA in mother-reared control rats. However, senescent maternally-deprived rats with impaired spatial learning ability showed increased expression of 5-HT_1A-receptor mRNA in the hippocampal CA1 (14%) and CA3 (13%) areas but not in the dentate gyrus.     

The 5-HT1A antagonist WAY 100635 can block the low-dose locomotor stimulant effects of cocaine

Cocaine treatments of 2.5, 5.0, 10.0 and 15.0 mg/kg induced dose-dependent increases in locomotor behavior. This cocaine-induced increase in locomotion was blocked if the animals were administered the selective 5-HT_1A antagonist, WAY 100635 (0.4 mg/kg) prior to the cocaine treatment. The 0.4-mg/kg dose of WAY 100635 did not affect locomotor behavior or alter cocaine availability in brain.     

Expression of 5-HT1A receptor mRNA in rat nucleus raphe magnus neurons after peripheral inflammation

The present study demonstrated that 5-HT_1A receptor mRNA was expressed with moderate level in the NRM neurons. Most of 5-HT_1A receptor mRNA positive cells were 5-HT neurons, suggesting the majority of 5-HT_1A receptor in the NRM might be autoreceptors. Eight hours after carrageenan inflammation, the expression of 5-HT_1A receptor mRNA in the NRM neurons, especially in the 5-HT neurons, was significantly increased. These results suggest that synthesis of 5-HT_1A receptors, including 5-HT_1A autoreceptors, is increased in the NRM during peripheral inflammation.     

Changes in rat cerebral blood volume due to modulation of the 5-HT1A receptor measured with susceptibility enhanced contrast MRI

Brain blood volume changes in the rat in response to 5-HT_1A agonist and antagonist administration were measured using susceptibility contrast enhanced magnetic resonance imaging (MRI). Administration of the 5-HT_1A agonist 8-OH-DPAT resulted in decreases in fractional brain blood volumes. Administration of the 5-HT_1A antagonist WAY-100635 following a dose of 8-OH-DPAT resulted in increases in fractional blood volumes greatest in hippocampus and cortex and smallest in thalamus and caudate-putamen. The magnitude of the regional increases in blood volumes paralleled the distribution of 5-HT_1A receptors in the rat brain. Administration of WAY-100635 alone resulted in decreases in cortical blood volume and increases in cerebellar blood volume.